This grant will support research designed to help answer the following questions: (1) What is the molecular mechanism of mammalian X-chromosome inactivation? (2) What is the biological function(s) of 5-methylcytosine in mammalian DNA? The working hypotheses that will guide this work are: (1) 5- Methylcytosine plays a significant role in mammalian gene regulation and cell differentiation, and (2) 5-Methylcytosine is an important component of the X-chromosome inactivation system. The specific aims are to: (1) identify and study proteins that bind specifically, in vitro and in vivo, to X-linked promoters and X-chromosomal DNA, (2) prepare and study DNA methylated at cytosine residues in CpG doublets by human DNA methyltransferase, (3) study proteins that bind preferentially to enzymatically methylated DNA, and (4) determine the effect of methylation on proteins identified as a result of aim 1.